Method for selecting polynucleotides based on enzyme interaction duration

ABSTRACT

A method for selecting polynucleotides, the method comprising: allowing a nucleic acid handling enzyme to move along multiple polynucleotides in a sample for a defined time period, wherein the enzyme is loaded onto each of the multiple polynucleotides and wherein one or more molecule of the enzyme moves along each of the multiple polynucleotides; and selecting polynucleotides based on whether or not the enzyme reaches the end of and/or unbinds from the polynucleotides in the defined time period.

RELATED APPLICATIONS

This Application is a national stage filing under 35 U.S.C. § 371 ofinternational application number PCT/GB2019/050029, filed Jan. 7, 2019,which claims the benefit of United Kingdom application number 1800187.5,filed Jan. 5, 2018, each of which is incorporated by reference in itsentirety.

FIELD

The invention relates generally to methods of selecting polynucleotides.The invention also relates generally to methods of modifying, separatingand/or characterising the selected polynucleotides.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 9, 2020, isnamed 0036670097US00-SUBSEQ-KZM and is 7 kilobytes in size.

BACKGROUND

Selection of polynucleotides based on size is important for manyapplications, such as sequencing DNA and RNA. There is a need for rapidand cheap polynucleotide characterisation, identification, amplificationand sequencing technologies across a wide range of applications. Thelength of the polynucleotide can aid in the identification of thepolynucleotide. The length and integrity of the polynucleotide can alsoaffect the success and rapidity of downstream identification,amplification or sequencing applications. Many high-throughput DNAsequencers require insert libraries of certain sizes for optimumperformance. Other examples of applications for which polynucleotidesize selection is important include ensuring correct PCR productformation, genotyping, DNA fingerprinting, gene profiling, andextracting a fragment of a defined size, for example following enzymaticdigestion of a polynucleotide.

DNA of different sizes is conventionally detected by gelelectrophoresis. Manual gel electrophoresis methods have many drawbacksand automated electrophoresis instruments that separate and select DNAof desired size ranges between 100 bp and 50 kb have been developed.

Functionalised, paramagnetic silica particles and polyethylene glycol(PEG) under different reaction conditions may be used for DNA sizeselection. Typically, these methods only allow the separation of DNA <1kb in length. Size exclusion methods using a solid matrix can also beused, but again, these are typically for the removal of short <1 kb DNAfragments from 1000 kb+ fragments.

SUMMARY

The inventors have devised an enzyme based method of selectingpolynucleotides in a sample comprising multiple polynucleotides. Theinventors have recognised that the speed of movement of nucleic acidhandling enzymes along the length of polynucleotides is consistent anddoes not depend on the length of the polynucleotide. The inventors haveused this property of nucleic acid handling enzymes to devise methods inwhich movement of a nucleic acid handling enzyme is used to selectpolynucleotides of different lengths. The method involves selectingpolynucleotides based on whether or not the enzyme remains bound to thepolynucleotides after a predetermined time period, and/or on whether ornot an enzyme reaches the end and/or falls off the polynucleotides. Themethod may be used to select polynucleotides of a desired length. Themethod may also be used to select undamaged polynucleotides and/orintact polynucleotides. Another use of the method is to selectpolynucleotides comprising adaptors at both ends from polynucleotidescomprising an adapter at only one end and/or from non-adaptedpolynucleotides.

Accordingly, provided herein is a method for selecting polynucleotides,the method comprising:

-   -   (i) allowing a nucleic acid handling enzyme to move along        multiple polynucleotides in a sample for a defined time period,        wherein the enzyme is loaded onto each of the multiple        polynucleotides and wherein one or more molecule of the enzyme        moves along each of the multiple polynucleotides; and    -   (ii) selecting polynucleotides based on whether or not the        enzyme reaches the end of and/or unbinds from the        polynucleotides in the defined time period.

The sample may, for example, comprise the products of a PCR reaction;genomic DNA; the products of a endonuclease digestion; or a DNA library.

The method may comprise one or more of the following:

-   -   selectively modifying polynucleotides of a desired length or        selectively modifying polynucleotides undesired lengths;    -   selectively modifying undamaged polynucleotides or selectively        modifying damaged polynucleotides;    -   selectively modifying intact polynucleotides or selectively        modifying nicked polynucleotides;    -   separating polynucleotides of a desired length from other        polynucleotides;    -   separating undamaged polynucleotides from damaged        polynucleotides;    -   separating intact polynucleotides from nicked polynucleotides;    -   characterising polynucleotides of a desired length, undamaged        polynucleotides and/or intact polynucleotides;    -   sequencing polynucleotides of a desired length, undamaged        polynucleotides and/or intact polynucleotides;    -   removing primers or adapters from polynucleotides of a desired        length, undamaged polynucleotides and/or intact polynucleotides;        and    -   genotyping, DNA fingerprinting or profiling using        polynucleotides of a desired length, undamaged polynucleotides        and/or intact polynucleotides.

Also provided are:

-   -   a method of characterising a polynucleotide, the method        comprising:    -   (i) carrying out a method as described herein;    -   (ii) contacting a transmembrane pore with the selected        polynucleotides;    -   (iii) applying a potential difference across the transmembrane        pore; and    -   (iv) taking one or more measurements which are indicative of one        or more characteristics of a polynucleotide moving with respect        to the transmembrane pore and thereby characterising the        polynucleotide;    -   a polymer adapter which has bound thereto:    -   (a) a first nucleic acid handling enzyme;    -   (b) a second nucleic acid handling enzyme, which is bound such        that its movement along the polymer is hindered until it is        brought into contact with a transmembrane pore, wherein the        second nucleic acid handling enzyme does not hinder movement of        the first nucleic acid handling enzyme; and optionally    -   (c) a membrane anchor or pore anchor; and    -   a kit for separating and/or selectively modifying        polynucleotides comprising (a) an adapter comprising a        polynucleotide and an end signal and/or (b) an adapter as        described above; and comprising any one or more, including any        combination, of the following components: an extraction medium;        a nucleic acid handling enzyme; a nucleotide that provides        energy for the enzyme, an enzyme cofactor and/or a coenzyme; a        solution comprising fuel and/or cofactor for the nucleic acid        handling enzyme; a wash solution, which does not contain fuel        and/or cofactor for the nucleic acid handling enzyme; a site        specific endonuclease; and/or a sequencing adapter.

DESCRIPTION OF THE FIGURES

It is to be understood that Figures are for the illustration purposesand are not intended to be limiting.

FIG. 1 shows a basic scheme where a nucleic acid handling enzyme isbound to a separating medium (e.g. beads), for example via a binding tag(e.g. a protein tag such as a his-tag or a strep-tag). (A) Enzymeadapter complexes bound to extraction media (e.g. beads). (B)Polynucleotides (e.g. double-stranded DNA) attached to adapters (e.g. byligation). (C) Fuel and cofactor added and system run for desiredperiod. The polynucleotides move relative to enzyme (the direction ofthe polynucleotides is indicated by arrows). The enzyme dissociates froma polynucleotide if it encounters a nick or gets to the end of a strand.If all enzymes are unbound the polynucleotide is capable of diffusinginto solution. The released polynucleotides can be prevented fromrebinding to free enzymes by various means. For example, trapoligonucleotides in solution can be designed to either bind and trap theenzymes, or bind and trap the free polynucleotide. Alternatively, theends of the adapters can be designed such that enzymes cannot load (e.g.there are no free single-stranded sites). (D) When the system isquenched, longer polynucleotide strands remain bound to enzyme, andshorter strands or damaged strands have dissociated. Unboundpolynucleotides (e.g. short strands or damaged strands) can be separatedfrom bound polynucleotides by conventional means, for example by washingbeads and optionally recovered. Bound polynucleotides (e.g. longerstrands) can be released and recovered in various ways. For example: theenzymes can be restarted by adding fuel and allowed to reach end ofstrands (step D back to step C); the enzymes can be unbound from thebeads (e.g. denaturing enzyme, inhibiting the tag binding, cleavingtag/enzyme, etc.); or the enzymes can be unbound from the DNA (e.g.denaturing, high salt, un-closing the closed-complex, etc.). For sizefraction selection, it is possible to recover any desired size fractionby looping between steps C>D>C>D . . . with loops of running for desiredtime>quenching>recovering elute from wash. For damaged strand selection,if after step D the long strands are eluted by re-adding fuel andrunning enzymes off the far ends of the polynucleotides, then anystrands with damage that causes the enzyme to get stuck (e.g. abasicregion, thymidine dimers, etc.) will remain bound to beads, and a can beseparated from unbound strands which were released by enzymes reachingtheir ends. For nick selection, strands with nicks will elute withshorter strands as the enzyme falls off before making it to the end.Therefore, recovery after step D for long strands also selects forstrands that contained no nicks (in at least one strand of a doublestranded polynucleotide if both ends were loaded with enzymes).

FIG. 2 shows a basic scheme where polynucleotides (e.g. double-strandedDNA) are bound to beads, for example via a binding tag, and an enzymethat reaches the far end of a polynucleotide can alter/displace the tag,and thus can be used to unbind the polynucleotide from the bead. Forsimplicity, this Figure shows an embodiment with two different adapterson a double stranded polynucleotide, one end with the enzyme and one endwith the binding tag. (A) DNA complexes bound to extraction media viatag (e.g. a biotinylated oligonucleotide binding to streptavidin coatedbeads). (B) Fuel and cofactor added and system run for desired period.Enzyme moves along the polynucleotides (the direction of enzyme movementis indicated by arrows). Enzyme dissociates from a polynucleotide if itencounters a nick or gets to the end of a strand. If enzyme reaches theend of a polynucleotide, it displaces the binding tag, allowing thepolynucleotide to dissociate from the bead. The released polynucleotidecan be prevented from rebinding to the tag in various ways, for exampleby the addition of excess capture strands in solution, which bind toeither the tag or the polynucleotide (Figure shows binding topolynucleotide). (C) When the system is quenched, long strands orstrands where enzyme could not reach the end (e.g. due to nicks) remainbound to the beads via the tag, and short strands are unbound. Unboundpolynucleotides (e.g. short strands) can be separated from boundnucleotides, for example by washing the beads, and optionally recovered.Bound polynucleotides (e.g. longer strands) can be released andrecovered in various ways. For example: the enzymes can be restarted byadding fuel and allowed to reach end of strands (effectively loopingback to step C); or the polynucleotides can be unbound from beads byother conventional means (e.g. unbinding tag, cleaving, elutionconditions such as changing pH/temperature, etc.). For size-fractionselection, it is possible to recover any desired size fraction bylooping between steps B>C>B>C . . . with loops of running for desiredtime>quenching>recovering elute from wash. For damaged strand selection,if after step C the long strands are eluted by re-adding fuel andrunning enzymes off far end, then any strands with damage that preventsthe enzyme reaching the end (e.g. abasic region, thymidine dimers,nicks, etc.) will remain bound to beads, and can be separated fromunbound strands where enzymes reached the end.

FIG. 3 shows examples of how selection criteria may be created using anexonuclease (A) or a polymerase (B). An exonuclease digests one strandof the polynucleotide. A polymerase will synthesise a complement, butotherwise can be used in a similar manner to a translocase. In theexamples shown in the Figure, the exonuclease and the polynucleotide aretagged and the tag is used to separate short polynucleotides from whichthe exonuclease or polymerase has dissociated from longerpolynucleotides to which the exonuclease or polymerase remains bound.

FIG. 4 shows examples of how a signal can be created if the enzymereaches the far end of the strand. FIG. 4A shows displacement of a tag.FIG. 4B shows how the enzyme can alter the far end of the adapter (c. byunzipping or displacing components), such as its structure. Thedifferences between ends that are altered versus intact ends can beexploited in subsequent attachment of adapters or components. Forexample, FIG. 4B illustrates how a structural change can be used to trapthe complementary hybridization site on the end of the strand to preventligation of a sequencing adapter, so that sequencing adapterspreferentially ligate to longer strands with intact ends. In thisillustration there is no requirement to separate the oligonucleotidesprior to sequencing-adapter attachments.

FIG. 5 shows an example of how the method may be implemented usingenzymes in solution, rather than, for example, pre-loaded onto anadapter that is attached to the polynucleotides in the sample. In thismethod the use of adapters is optional, enzymes may instead bindnaturally to the polynucleotides in the sample, either at the ends or tothe middle of the strands. The enzyme is allowed to bind freely to thepolynucleotides, optionally under conditions where the enzyme is free tomove along the polynucleotides, e.g. in the presence of fuel such asATP. Allowing the enzyme to bind in this manner will result in theeventual saturation of the strands by the enzyme. After enzyme has beenallowed to bind to the polynucleotides, a defined time period is startedby removing enzymes from solution and/or adding a large excess ofcapture strand to prevent additional molecules of enzyme from binding tothe polynucleotides. The enzyme typically binds preferentially to thecapture strand over the polynucleotides in the samples. At the end ofthe defined time period, enzyme movement is stopped, typically byquenching, and polynucleotides to which enzymes remain bound areseparated from shorter polynucleotides to which enzymes are no longerbound.

FIG. 6 shows a further example of how the method may be implementedusing enzymes in solution. In this method tagged adapters are used. Adefined time period is started by contacting the polynucleotides withthe enzyme in solution containing the cofactors and fuel necessary forthe enzyme to move along the polynucleotides. In this example, theenzymes are only able to load onto the ends of the polynucleotides inthe sample via the attached adapter. For clarity the Figure only showsenzymes loading onto the single-stranded overhang of the left adapterand running along the top strand of the polynucleotides. Enzymes thatreach the end of the polynucleotides displace a selection tag in theadapter at the end of the polynucleotide. At the end of the time periodenzyme movement is stopped and polynucleotides which retain theselection tag are separated from shorter polynucleotides from which theselection tag has been displaced.

FIG. 7 shows examples of polynucleotides that can be separated by themethods. The polynucleotide may comprise a hairpin adapter at one orboth ends. Single stranded polynucleotides or double strandedpolynucleotides may be separated by the method.

FIG. 8 shows one particular example of how the method may be used toselect and prepare polynucleotides for characterisation using atransmembrane pore using a dual purpose adapter that comprises (1) atagged enzyme for size selection; and (2) a leader sequence, a stalledenzyme and a membrane tether for nanopore sequencing. FIG. 8A shows anadapter that may be used in the separation method and then to facilitatecharacterisation of selected polynucleotides using a transmembrane pore.The adapter comprises a stalled nucleic acid handling enzyme and anun-stalled tagged enzyme. FIG. 8B shows how the adapter may be attachedto polynucleotides prior to adding fuel to start movement of theun-stalled tagged enzyme along the polynucleotides. At the end of adefined time period, the movement of the tagged enzyme is stopped andlonger polynucleotides to which the tagged enzyme is still bound areseparated from shorter polynucleotides from which the tagged enzyme hasbeen released. All of the polynucleotides retain an adapter having anenzyme stalled attached. Therefore, the polynucleotides of interest(which may be the longer polynucleotides or the shorter polynucleotides)are in a form that can immediately be sequenced using a transmembranepore. The stalled enzyme serves to control the movement of thepolynucleotides through a transmembrane pore. The stall is overcome whenthe stalled enzyme comes into contact with the transmembrane pore in thepresence of fuel.

FIG. 9 shows the size distribution of polynucleotides selected from alibrary of DNA of mixed lengths to which an adapter with an un-stalledhelicase had been bound. The size of the polynucleotides was determinedby nanopore sequencing. ATP and Mg were added to the library for 120seconds or 240 seconds prior to stopping the helicase and selecting theDNA strands to which the helicase remained bound. FIG. 9A compares thestarting size distribution of the initial library (0 s incubation time)with the size distribution of the DNAs selected from the library afterallowing the helicase to run for 240 seconds. FIG. 9B shows thedistributions as binned histograms in separate panels. The histogramsclearly show the reduction in strand counts for shorter strands in the1-3 kilobases range, and a relative increase in strand count for strandsgreater than approximately 3 kilobases in length for the libraries with120 s or 240 s incubation times.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is the nucleotide sequence of the top strand of the sizeselection adapter used in Examples 1 and 2.

SEQ ID NO: 2 is the nucleotide sequence of the bottom strand of the sizeselection adapter used in Examples 1 and 2.

SEQ ID NO: 3 is the nucleotide sequence of the blocker strand of thesize selection adapter used in Example 1.

SEQ ID NO: 4 is the amino acid sequence of the strep-SUMO tag attachedto the DNA helicase used in Examples 1 and 2.

SEQ ID NO: 5 is the nucleotide sequence of the 3.6 kb DNA used inExample 1.

SEQ ID NO: 6 is the nucleotide sequence of the blocker strand of thesize selection adapter used in Example 2.

It is to be understood that sequences are not intended to be limiting.

DETAILED DESCRIPTION

It is to be understood that different applications of the disclosedmethods and products may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the methods and productsonly, and is not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “ananchor” refers to two or more anchors, reference to “a helicase”includes two or more helicases, and reference to “a transmembrane pore”includes two or more pores and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The present inventors have devised a method for selectingpolynucleotides, the method comprising:

-   -   (i) allowing a nucleic acid handling enzyme to move along        multiple polynucleotides in a sample for a defined time period,        wherein the enzyme is loaded onto each of the multiple        polynucleotides and wherein one or more molecule of the enzyme        moves along each of the multiple polynucleotides; and    -   (ii) selecting polynucleotides based on whether or not the        enzyme reaches the end of and/or unbinds from the        polynucleotides in the defined time period.

The method may further comprise separating the selected polynucleotidesfrom the non-selected polynucleotides and/or selectively modifying theselected polynucleotides or the non-selected polynucleotides. Typically,the enzyme is loaded onto each of the multiple polynucleotides in thesame manner.

Nucleic Acid Handling Enzyme

The method utilizes a nucleic acid handling enzyme to enablepolynucleotides with different physical properties to be selectivelymodified and/or separated. The nucleic acid handling enzyme may be usedas a marker to tag polynucleotides within a sample that meet theselection criteria, or alternatively to tag polynucleotides in thesample that do not meet the selection criteria. In another embodiment,the action of the nucleic acid handling enzyme alters a signal at theterminal end of the polynucleotide enabling that signal to be used toseparate polynucleotides that meet the selection criteria frompolynucleotides that do not meet the selection criteria. The signal atthe terminal end of the polynucleotide may, for example, be a hiddensignal that is exposed when the enzyme reaches the terminal end of thepolynucleotide. Alternatively, the signal may be an exposed signal thatis removed when the enzyme reaches the terminal end of thepolynucleotide.

The nucleic acid handling enzyme may be any protein that is capable ofbinding to a polynucleotide and processing the polynucleotide. Inprocessing the polynucleotide, the nucleic acid handling enzyme movesalong the polynucleotide. The direction of movement of the enzyme isconsistent. Consistent movement means that the enzyme moves from the 5′end to the 3′ end of the polynucleotide or vice versa. The enzyme maymodify the polynucleotide as it processes it. It is not essential thatmodification of the polynucleotide occurs. Therefore, the nucleic acidhandling enzyme may be a modified enzyme that retains its ability tomove along a polynucleotide.

The nucleic acid handling enzyme may be, for example, a translocase, ahelicase, a polymerase or an exonuclease.

The nucleic acid handling enzyme may move along a single strandedpolynucleotide, such as single stranded DNA or single stranded RNA, ormay move along a double stranded polynucleotide such as double strandedDNA or a DNA/RNA hybrid. For example, helicases or translocases that acton either single stranded or double stranded DNA may be used.

The helicase may, for example, be a member of superfamily 1 orsuperfamily 2.

The helicase is preferably a member of one of the following families:Pif-like, Upf1-like, UvrD/Rep, Ski-like, Rad3/XPD, NS3/NPH-II, DEAD,DEAH/RHA, RecG-like, REcQ-like, T1R-like, Swi/Snf-like and Rig-I-like.The first three of those families are in superfamily 1 and the secondten families are in superfamily 2. The helicase is more preferably amember of one of the following subfamilies: RecD, Upf1 (RNA), PcrA, Rep,UvrD, Hel308, Mtr4 (RNA), XPD, NS3 (RNA), Mss116 (RNA), Prp43 (RNA),RecG, RecQ, T1R, RapA and Hef (RNA). The first five of those subfamiliesare in superfamily 1 and the second eleven subfamilies are insuperfamily 2. Members of the Upf1, Mtr4, NS3, Mss116, Prp43 and Hefsubfamilies are RNA helicases. Members of the other subfamilies are DNAhelicases.

The helicase may be a multimeric or oligomeric helicase. In other words,the helicase may need to form a multimer or an oligomer, such as adimer, to function. The helicase is preferably monomeric. In otherwords, the helicase preferably does not need to form a multimer or anoligomer, such as a dimer, to function. For example, He308, RecD, TraIand XPD helicases are all monomeric helicases. These are discussed inmore detail below. Methods for determining whether or not a helicase isoligomeric/multimeric or monomeric are known in the art. For instance,the kinetics of radiolabelled or fluorescently-labelled polynucleotideunwinding using the helicase can be examined. Alternatively, thehelicase can be analysed using size exclusion chromatography.

Monomeric helicases may comprise several domains attached together. Forinstance, TraI helicases and TraI subgroup helicases may contain twoRecD helicase domains, a relaxase domain and a C-terminal domain. Thedomains typically form a monomeric helicase that is capable offunctioning without forming oligomers.

Particular examples of suitable helicases include Hel308, NS3, Dda,UvrD, Rep, PcrA, Pif1 and TraI. These helicases typically work on singlestranded DNA. Examples of helicases that can move along both strands ofa double stranded DNA include FtfK and hexameric enzyme complexes, ormultisubunit complexes such as RecBCD.

The helicase may, for example, be any of the helicases, modifiedhelicases or helicase constructs disclosed in WO 2013/057495, WO2013/098562, WO2013098561, WO 2014/013260, WO 2014/013259, WO2014/013262 and WO/2015/055981. The Hel308 helicase preferably comprisesany one or more of the modifications disclosed in WO 2014/013260. TheDda helicase preferably comprises any one or more of the modificationsdisclosed in WO 2015/055981 and/or WO 2016/055777.

The nucleic acid handling enzyme may be a polymerase. A polymerase willtypically synthesize a complementary polynucleotide strand as it movesalong a polynucleotide. Otherwise, a polymerase may be used in a similarmanner to a translocase. The polymerase may be a modified polymerasewhich retains its ability to move along a polynucleotide, but which doesnot synthesize a complementary strand. The polymerase may, for example,be PyroPhage® 3173 DNA Polymerase (which is commercially available fromLucigen® Corporation), SD Polymerase (commercially available fromBioron®) or variants thereof. The enzyme is preferably Phi29 DNApolymerase or a variant thereof.

Synthesis of a complementary strand may be advantageous in that itincreases the amount of polynucleotide. Increasing the amount ofpolynucleotide may improve sensitivity of any subsequent assay using thepolynucleotide selected by the method. Where the polynucleotide containsmodified bases, the polymerase may be used to synthesize a complementarystrand that contains normal bases, which can also be advantageous forsubsequent assays using the polynucleotide.

Using a polymerase may have the advantage that it can be used todistinguish a damaged polynucleotide from an undamaged polynucleotide.For example, the polymerase may be unable to pass through an abasicnucleotide in DNA or through thymidine dimers. Accordingly, a methodusing a polymerase may be used to separate damaged polynucleotides fromundamaged polynucleotides.

The nucleic acid handling enzyme may be an exonuclease. An exonucleasetypically digest the polynucleotide as it moves along it. Theexonuclease typically cleaves one strand of a double strandedpolynucleotide to form individual nucleotides or shorter chains ofnucleotides, such as di- or tri-nucleotides. Where an exonuclease isused, the polynucleotides which are ultimately selected are theundigested strands of double stranded polynucleotide, or polynucleotidesin which one of the strands is partially digested and the other strandis intact. Any exonuclease enzyme may be used in the method. Preferredenzymes for use in the method include exonuclease III enzyme from E.coli, exonuclease I from E. coli, bacteriophage lambda exonuclease andenzymes derived from exonuclease III enzyme from E. coli, exonuclease Ifrom E. coli, bacteriophage lambda exonuclease. An enzyme derived fromone of these exonucleases preferably comprises the domains responsiblefor binding to the nucleic acid and for digesting the nucleic acid(catalytic domain).

The nucleic acid handling enzyme is preferably one that is able toprocess long polynucleotide strands without unbinding from thepolynucleotide. Typically, the nucleic acid handling enzyme is capableof moving along a polynucleotide strand of from 500 nucleotide basepairs up to 250 million nucleotide base pairs, such as from 1,000,2,000, 5,000, 10,000, 50,000 or 100,000 nucleotide base pairs up to 200million, 100 million, 10 million or 1 million nucleotide base pairs.

The enzyme may be modified or unmodified. The enzyme may be modified toform a closed-complex. A closed-complex is an enzyme in which thepolynucleotide binding site is modified such that the enzyme is closedaround the polynucleotide in such a way that the enzyme does not falloff the polynucleotide other than when it reaches the end of thepolynucleotide. Examples of suitable closed-complex enzymes and methodsfor modifying enzymes to produce closed complexes are disclosed in, forexample, WO 2014/013260 and WO 2015/055981.

Where the nucleic acid handling enzyme is an unmodified polymerase, theenzyme is typically capable of moving along a polynucleotide of up to 30kb. The distance of movement may be increased by modifying thepolymerase to close an opening from which the polynucleotide is able tounbind when the enzyme is part way along the polynucleotide. For such amodified polymerase, the longer polynucleotide lengths specified abovemay be processed by the polymerase.

During step (i) of the method, one molecule of the enzyme may move alongeach of the multiple polynucleotides. In alternative embodiments,multiple molecules of the enzyme may move along each of the multiplepolynucleotides. The number of molecules of the enzyme moving along eachof the multiple polynucleotide will depend on the method of loading theenzyme onto the polynucleotides.

Where multiple molecules of the enzyme move along the multiplepolynucleotides, the exact number of molecules is not important. Forexample, at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10molecules of the enzyme may move along a polynucleotide. All that isrequired for a method which uses multiple molecules of an enzyme on eachof the multiple polynucleotides is that at the end of the defined timeperiod, polynucleotides to which one or more molecule of the enzymeremains bound can be separated from polynucleotides to which nomolecules of the enzyme remain bound, or alternatively thatpolynucleotides which have had at least one molecule of the enzyme passthe terminal end can be separated from polynucleotides where none of themolecules of bound enzyme have reached the terminal end. In thesemethods, the enzyme is added in solution either prior to step (i) or toinitiate the defined time period in step (i). Binding enzymes fromsolution has the advantage that it simplifies sample preparation. Wherethe enzyme is in solution, binding of the enzyme to the start of thepolynucleotide can occur freely. Binding of the enzyme in solution tothe initial polynucleotides may be stopped or prevented by sequesteringfree enzyme in solution, such as, for example, by adding a capturepolynucleotide (capture strand) or other capture molecule, such asheparin. A capture polynucleotide or molecule is preferentially bound bythe free enzyme, due to its higher relative affinity for the freeenzyme, than the polynucleotide that is being characterised/the targetpolynuclotide. The higher affinity can be achieved by providing a thecapture polynucleotide or molecule at a higher concentration than thepolynucleotide being characterised.

A capture strand is typically a short strand of DNA or RNA to whichhelicase binds. The capture strand may have a length of from about 15nucleotides to about 40 nucleotides, such as about 20, about 25 or about30 nucleotides. The capture strand is typically added at a highconcentration in solution to promote binding of helicase in solution tothe capture strand in preference to the polynucleotides being separated.The skilled person will readily be able to identify a suitableconcentration of capture strand. The concentration may, for example, befrom about 10 nM to about 1M, such as from about 50 nM to about 500 mMor about 1 mM to 100 mM.

FIGS. 5 and 6 show schematic examples of how the method can utiliseenzymes in solution. Where the enzyme is in solution, more enzymes willtypically continue to bind. Therefore, the selection is not based on theuse of a single enzyme. Selection can be based on enzyme reaching end tocreate or destroy a signal (FIG. 6). Selection can be based on taggedenzymes (FIG. 5). For simplicity, binding from only one end of thestrand is shown in FIGS. 5 and 6.

Loading Enzyme onto Polynucleotide

In one embodiment, the method comprises an initial step of binding theenzyme to the multiple polynucleotides. Thus, at the start of step (i)one or more molecules of the enzyme may be bound to each of the multiplepolynucleotides. Only one molecule of enzyme may be bound to each of themultiple polynucleotides at the start of step (i) or multiple moleculesof the enzyme may be bound to each of the multiple polynucleotides atthe start of step (i).

Where only one molecule of enzyme is bound to each of the multiplepolynucleotides, the enzyme is typically pre-bound to an adapter. Theinitial step of binding the enzyme to the multiple polynucleotides maytherefore comprise attaching an adapter having an enzyme bound theretoto one or both ends of the multiple polynucleotides.

Where multiple molecules of the enzyme are bound to each of the multiplepolynucleotides prior to step (i), the enzymes are added to thepolynucleotides in solution during the initial step of binding theenzyme to the multiple polynucleotides. In this embodiment, adapters mayalso be attached to the polynucleotides prior to addition of the enzymein solution. The use of an adapter is not essential in this embodiment.Many enzymes, helicases in particular, can bind to genomic DNA with arange of available ends. Typically, in an embodiment which uses no endadapters, the nucleic acid handling enzyme comprises a selection tag,e.g. a selection tag is bound to the enzyme. Where an adapter is used,the adapter may contain, for example, a poly T loading site for ahelicase, or for two or more helicases. The adapter may be designed forenzymes that move in the 5′-3′ or 3′-5′ direction as required.

In an alternative embodiment, at the start of step (i) there may be nomolecules of the enzyme bound to any of the multiple polynucleotides. Inthis embodiment, the defined time period is started by adding the enzymeto the sample in solution. Any fuel, coenzymes or cofactors necessaryfor movement of the enzyme may be added prior to, or together with theenzyme.

Sample

The sample may be any suitable sample comprising polynucleotides. Thepolynucleotides may, for example, comprise the products of a PCRreaction, genomic DNA, the products of a endonuclease digestion and/or aDNA library.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaean,prokaryotic or eukaryotic and typically belongs to one the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus.

The sample is preferably a fluid sample. The sample typically comprisesa body fluid. The body fluid may be obtained from a human or animal. Thehuman or animal may have, be suspected of having or be at risk of adisease. The sample may be urine, lymph, saliva, mucus, seminal fluid oramniotic fluid, but is preferably whole blood, plasma or serum.Typically, the sample is human in origin, but alternatively it may befrom another mammal such as from commercially farmed animals such ashorses, cattle, sheep or pigs or may alternatively be pets such as catsor dogs.

Alternatively a sample of plant origin is typically obtained from acommercial crop, such as a cereal, legume, fruit or vegetable, forexample wheat, barley, oats, canola, maize, soya, rice, bananas, apples,tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa,cotton, tea or coffee.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of non-biological samples includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample may be processed prior to carrying out the method, forexample by centrifugation or by passage through a membrane that filtersout unwanted molecules or cells, such as red blood cells. The method maybe performed on the sample immediately upon being taken. The sample mayalso be typically stored prior to the method, preferably below −70° C.

The sample may comprise genomic DNA. The genomic DNA may be fragmented.The DNA may be fragmented by any suitable method. For example, methodsof fragmenting DNA are known in the art, Such methods may use atransposase, such as a MuA transposase. Preferably the genomic DNA isnot fragmented.

The polynucleotides may be unmodified. Adapters may be added to one orboth ends of the polynucleotides in the sample. Suitable adapters aredefined below.

In one embodiment, a hairpin adapter may be added to one or both ends ofthe polynucleotides. Where a hairpin adapter is added to both ends ofthe polynucleotides, an enzyme is preferably prebound to the adapters.In this embodiment, the hairpins will typically prevent the enzyme fromfalling off the ends of the polynucleotides. Therefore, the adapterspreferably comprise a signal that is removed or activated when an enzymemoves from the adapter at one end of the polynucleotide to the adapterat the other end of the polynucleotide. Examples of embodiments usinghairpin adapters are shown in FIG. 7.

Multiple Polynucleotides

The disclosed method is used to select polynucleotides from a samplecomprising multiple polynucleotides. The term “multiple” is used hereinto mean two or more different polynucleotides, such as from about atleast 2, at least 3 or at least 4 to about 100,000 or more, for examplefrom about at least 5 to about 50,000 or more, about at least 10 toabout 10,000 or more polynucleotides.

In certain exemplary embodiments, the sample may comprise at least about20, at least about 50, at least about 100, at least about 500 or atleast about 1,000 different polynucleotides.

The polynucleotides separated by the method may be, for example, DNA,RNA and/or DNA/RNA hybrids. The DNA may be double stranded or singlestranded. The sample may comprise different polynucleotides of the sametype, such as, for example, different DNAs, different RNAs or differentRNA/DNA hybrids. The sample may comprise multiple types ofpolynucleotide such as any two or more of DNA, RNA and DNA/RNA hybrids.

The polynucleotide can be in any suitable form. Adapters may be added toone or both ends of the polynucleotides. The adapters may, for example,be used to ensure equal loading of the enzyme onto differentpolynucleotides within a sample and/or to ensure that thepolynucleotides in the sample are in the same form. Any suitable adapterdesign may be used. For example the adapters may be designed for singleor double-ended attachment.

Adapters

In an embodiment of the method, an adapter may be attached to one orboth ends of each of the multiple polynucleotides. The method maycomprise an initial step of attaching an adapter to one or both ends ofthe multiple polynucleotides. The initial step may further comprisebinding the enzyme to the adapter. Alternatively, the enzyme may bepre-bound to the adapter. Thus, the method may comprise an initial stepof attaching an adapter having the enzyme pre-bound thereto to one orboth ends of each of the multiple polynucleotides under conditions wherethe enzyme does not move along the polynucleotides. The enzyme may bestalled on the adapter. The adapter may be stalled by virtue of theabsence of fuel and/or a necessary cofactor. The adapter may be stalledin the presence of fuel, using a stall that can be removed/overcome toinitiate movement of the enzyme (e.g. by toehold displacement).

The same adapter may be added to both ends of the multiplepolynucleotides. Alternatively, different adapters may be added to thetwo ends of each of the multiple polynucleotides. An adapter may beadded to just one end of each of the multiple polynucleotides. Methodsof adding adapters to polynucleotides are known in the art. Adapters maybe attached to polynucleotides, for example, by ligation, by clickchemistry, by tagmentation, by topoisomerisation or by any othersuitable method.

The adapter is preferably capable of being attached to the end of apolynucleotide to which a nucleic acid handling enzyme can bind. Theadapter is preferably synthetic or artificial. The adapter preferablycomprises a polymer. The polymer is preferably a polynucleotide. Thepolynucleotide adapter may comprise DNA, RNA, modified DNA (such as abasic DNA), RNA, PNA, LNA, BNA and/or PEG. The adapter more preferablycomprises single stranded and/or double stranded DNA or RNA. Thepolynucleotide may be of any suitable length, for example from about 4to about 300, such as about 5 to about 200, about 10 to about 100, orabout 20 to about 50 nucleotides in length.

The adapter may comprise a single stranded polynucleotide to which thenucleic acid handling enzyme is bound.

The adapter used for selection may be designed to facilitate thesubsequent attachment of selected polynucleotides to further adapters,such as sequencing adapters. The adapter may, for example, comprise asingle-stranded overhang or chemical group (e.g. click chemistry) forefficient attachment to a further adapter.

In one embodiment, the adapter is a Y adapter. A Y adapter and/or thebridging moiety adapter are typically polynucleotide adapters. A Yadapter is typically double stranded and comprises (a) at one end, aregion where the two strands are hybridised together and (b), at theother end, a region where the two strands are not complementary. Thenon-complementary parts of the strands form overhangs. The presence of anon-complementary region in the Y adapter gives the adapter its Y shapesince the two strands typically do not hybridise to each other unlikethe double stranded portion. A nucleic acid handling enzyme may be boundto an overhang and/or to the double stranded region. In one embodiment,a first enzyme is bound to the double stranded region and a secondenzyme is bound to an overhang. The second enzyme on the overhang ispreferably stalled by a spacer. In one embodiment the Y adaptercomprises a membrane anchor or a pore anchor. The anchor may be attachedto a polynucleotide that is complementary to and hence that ishybridised to the overhang which an enzyme is not bound.

One of the non-complementary strands Y adaptor typically comprises aleader sequence, which when contacted with a transmembrane pore iscapable of threading into the pore. The leader sequence typicallycomprises a polymer. The polymer is preferably negatively charged. Thepolymer is preferably a polynucleotide, such as DNA or RNA, a modifiedpolynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG)or a polypeptide.

The leader preferably comprises a polynucleotide and more preferablycomprises a single stranded polynucleotide. The single stranded leadersequence most preferably comprises a single strand of DNA, such as apoly dT section. The leader sequence preferably comprises the one ormore spacers.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 120, 30 to 100, 40 to 80 or 50to 70 nucleotides in length.

In one embodiment the adapter is a hairpin loop adapter. A hairpin loopadapter is an adapter comprising a single polynucleotide strand, whereinthe ends of the polynucleotide strand are capable of hybridising to eachother, or are hybridized to each other, and wherein the middle sectionof the polynucleotide forms a loop. Suitable hairpin loop adapters canbe designed using methods known in the art. The loop may be any length.The loop is preferably from about 2 to 400, from 5 to 300, from 10 to200, from 20 to 100 nucleotides or from 30 to 50 in length. The doublestranded section of the adapter formed by two hybridized sections of thepolynucleotide strand is called a stem. The stem of the hairpin loop ispreferably from 4 to 200, such as 5 to 150, 10 to 100, 20 to 90, 30 to80, 40 to 70 or 50 to 60 nucleotide pairs in length. Where a nucleicacid handling enzyme is bound to or binds to a hairpin adapter, ittypically binds to the loop of the hairpin, rather than to the stem.

If the multiple polynucleotides are double stranded, a Y adapter may beadded to one end and a hairpin loop adapter to the other end. In thisembodiment, an enzyme may be bound to the Y adapter and/or to thehairpin adapter.

The adapter may comprise a second nucleic handling enzyme, preferably ahelicase that is stalled on the adapter, for example by or at a spacer.

The adapters may be attached to the multiple polynucleotides in anymanner. The adapters are preferably covalently attached to the targetpolynucleotide.

The adapters may be ligated to the target polynucleotide. The adaptersmay be ligated to either end of the polynucleotide, i.e. the 5′ or the3′ end, or to both ends of the polynucleotide i.e. to the 5′ end and tothe 3′ end. The adapters may be ligated to the polynucleotide using anymethod known in the art. The adapter may be ligated to thepolynucleotides in the absence of ATP or using gamma-S-ATP (ATPγS)instead of ATP. It is preferred that the adapter is ligated to thepolynucleotides in the absence of ATP where the nucleic acid handlingenzyme is bound to the adapter.

The adapter may be ligated using a ligase, such as T4 DNA ligase, E.coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase. Theligase may be removed from the sample before step (i) of the method. Theadapter may be attached using a topoisomerisase. The topoisomerase may,for example be a member of any of the Moiety Classification (EC) groups5.99.1.2 and 5.99.1.3.

The inventors have devised an adapter which has bound thereto: a firstnucleic acid handling enzyme; and a second nucleic acid handling enzyme,wherein the second nucleic acid enzyme is bound such that its movementalong the adapter is hindered or prevented until it is brought intocontact with a transmembrane pore under an applied potential, andwherein the second nucleic acid handling enzyme does not hinder movementof the first nucleic acid handling enzyme. In this embodiment, theadapter preferably comprises a polynucleotide and/or the second nucleicacid enzyme is preferably a translocase or helicase. The first andsecond nucleic acid handling enzymes may be the same or different. Forexample, the first enzyme may be a translocase, helicase or polymeraseand the second enzyme may be a translocase or helicase. Where both thefirst and second enzymes are both translocases or helicases, they may bethe same or different translocases or helicases.

Movement of the second enzyme may be hindered or prevented by beingstalled at a spacer, for example as disclosed in WO 2014/135838. Anyconfiguration of enzymes and spacers disclosed in WO 2014/135838 may beused in the method of separating polynucleotides.

The spacer is preferably part of the adapter, for instance the spacermay interrupt the polynucleotide sequence in the adapter. There may beany number of spacers in the adapter, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more spacers. There are preferably one, two, four or six spacersin the target polynucleotide. The one or more spacers are preferablylocated between the first and second enzymes on the adapter. The firstenzyme is preferably on the side of the spacer that is towards the endof the adapter that is attached to, or is for attachment to, apolynucleotide. The spacer is preferably positioned between the secondenzyme and the end of the adapter that is attached to, or is forattachment to, a polynucleotide. Alternatively, the second enzyme may bepositioned on the spacer.

The spacer provides an energy barrier which the second enzyme cannotovercome even in the presence of fuel and the necessary coenzymes and/orcofactors. The spacer may stall the second enzyme by reducing thetraction of the enzyme (for example the bases from the nucleotides inthe spacer may be missing) or by physically blocking movement of the oneor more helicases (for example, the spacer may comprise a bulky chemicalgroup).

The spacer may comprise any molecule or combination of molecules thathinders or prevents the second enzyme from moving along the targetpolynucleotide. It is straightforward to determine whether or not anenzyme is stalled at a spacer, in the absence of a transmembrane poreand an applied potential. For example, the ability of an enzyme to movepast a spacer and displace a complementary strand of DNA can be measuredby polyacrylamide gel electrophoresis (PAGE).

The spacer typically comprises a linear molecule, such as a polymer. Thespacer typically has a different structure from the targetpolynucleotide. For instance, if the target polynucleotide is DNA, theone or more spacers are typically not DNA. In particular, if the targetpolynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),the spacer preferably comprise peptide nucleic acid (PNA), glycerolnucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid(LNA) or a synthetic polymer with nucleotide side chains. The spacer maycomprise one or more nucleotides in the opposite direction from othernucleotides in the adapter. For example, the spacer may comprise one ormore nucleotides in the 3′ or 5′ direction when the polynucleotide is inthe 5′ to 3′ direction.

The spacer preferably comprises one or more nitroindoles, such as5-nitroindoles, inosines, acridines, 2-aminopurines, 2-6-diaminopurines,5-bromo-deoxyuridines, inverted thymidines (inverted dTs), inverteddideoxy-thymidines (ddTs), dideoxy-cytidines (ddCs), 5-methylcytidines,5-hydroxymethylcytidines, 2′-O-Methyl RNA bases, Iso-deoxycytidines(Iso-dCs), Iso-deoxyguanosines (Iso-dGs), iSpC3 groups (i.e. nucleotideswhich lack sugar and a base), photo-cleavable (PC) groups, hexandiolgroups, spacer 9 (iSp9) groups, spacer 18 (iSp18) groups, a polymer orthiol connections. The spacers may comprise any combination of thesegroups. Many of these groups are commercially available from IDT®(Integrated DNA Technologies®).

The spacer may contain, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more 2-aminopurines, 2-6-diaminopurines, 5-bromo-deoxyuridines,inverted dTs, ddTs, ddCs, 5-methylcytidines, 5-hydroxymethylcytidines,2′-O-Methyl RNA bases, Iso-dCs, Iso-dGs, iSpC3 groups, PC groups,hexandiol groups and thiol connections, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or more. The spacer preferably comprises 2, 3, 4, 5, 6, 7, 8 or moreiSp9 groups and/or 2, 3, 4, 5 or 6 or more iSp18 groups. The mostpreferred spacer is four iSp18 groups.

Where the spacer comprises a polymer, the polymer is preferably apolypeptide or a polyethylene glycol (PEG). The polypeptide preferablycomprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. ThePEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moremonomer units.

The spacer may comprise one or more abasic nucleotides (i.e. nucleotideslacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore abasic nucleotides. The nucleobase can be replaced by —H (idSp) or—OH in the abasic nucleotide. Abasic spacers can be inserted into targetpolynucleotides by removing the nucleobases from one or more adjacentnucleotides. For instance, polynucleotides may be modified to include3-methyladenine. 7-methylguanine, 1,N6-ethenoadenine inosine orhypoxanthine and the nucleobases may be removed from these nucleotidesusing Human Alkyladenine DNA Glycosylase (hAAG). Alternatively,polynucleotides may be modified to include uracil and the nucleobasesremoved with Uracil-DNA Glycosylase (UDG). In one embodiment, the one ormore spacers do not comprise any abasic nucleotides.

The second enzyme may be stalled before or on a linear molecule spacer.If a linear molecule spacer is used, the adapter preferably comprises adouble stranded region of polynucleotide adjacent to the end of thespacer closest to the end of the adapter which is attached to, or is forattachment to, the polynucleotide. A hybridized double stranded regionpreferably terminates at the spacer and the strand that does notcomprise the spacer preferably forms an overhang adjacent to the spacer.A further polynucleotide strand may be hybridized to the overhang toform a further double stranded region. The further double strandedregion typically helps to stall the second enzyme on the spacer. Thefurther polynucleotide is typically formed from the same nucleotides asthe target polynucleotide, but may be formed from different nucleotides.For instance, the further polynucleotide may be formed from lockednucleic acid (LNA) or bridged nucleic acid (BNA).

If a linear molecule spacer is used, the adapter preferably comprises ablocking molecule at the end of the spacer. The blocking molecule mayhelp to ensure that the second enzyme remains stalled on the spacer. Theblocking molecule may be any chemical group which physically causes theone or more helicases to stall. The blocking molecule may be a doublestranded region of polynucleotide. Examples of suitable blockingpolynucleotides are disclosed in the Examples.

Suitable chemical groups include pendant chemical groups. The chemicalgroup may be attached to one or more nucleobases in the targetpolynucleotide and/or to the polynucleotide backbone. Any number ofchemical groups may be present, such as 1, 2, 3. 4, 5, 6, 7, 8, 9, 10,11, 12 or more. Examples of suitable groups include, but are not limitedto, fluorophores, streptavidin and/or biotin, cholesterol, methyleneblue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin anddibenzylcyclooctyne groups.

Where more than one spacer is present in an adapter they may be the sameor different. For example, one spacer may comprise one of the linearmolecules discussed above and another spacer may comprise one or morechemical groups which physically stall the second enzyme. A spacer maycomprise any of the linear molecules discussed above and one or morechemical groups. such as one or more abasics and a fluorophore.

Most nucleic acid handling enzymes, such as helicases, bind and movealong DNA and so may be stalled using anything that is not DNA.

In the absence of a transmembrane pore and an applied potential, thespacer is preferably capable of stalling the second enzyme in thepresence of free nucleotides and/or the presence of a cofactor.

Step (i) of the method of separating polynucleotides is carried out inthe presence of fuel (free nucleotides) and a cofactor. The length andcharacteristics of the spacer are typically chosen to ensure that theone or more helicases are stalled on the adapter during the method.

The ability of a spacer to stall an enzyme may be affected by saltconcentration. The higher the salt concentration used in the method ofthe invention, the shorter the one or more spacers need to be. In theabsence of a transmembrane pore and an applied potential, the spacer ispreferably capable of stalling the second-enzyme at a salt concentrationof less than about 100 mM.

Examples of spacers that can be used to stall an enzyme that process DNAin the presence of free nucleotides and a cofactor include: at 1M salt,4 iSpC3 groups or 2 iSp18 groups; at 100-1000 mM salt, 4 iSp18 groups or6 iSp9 groups; at <100-1000 mM salt, 6 iSp18 groups, 12 iSpC3 groups or20 iSpC3 groups.

An enzyme can be ‘pushed over’ a stalling chemistry by annealing a DNAstrand behind the enzyme, e.g. by toehold displacement. Alternatively,an enzyme can be ‘stalled’ using a condition in which it is unable totranslocate. For example, the adapter may be kept in a pH at which theenzyme is unable to translocate and/or bind fuel. A small moleculeinhibitor could alternatively be used to stall an enzyme.

In one embodiment, the adapter further comprises a membrane anchor orpore anchor. The anchor is preferably present when a first enzyme and asecond stalled enzyme are bound to the adapter. Suitable anchors areknown in the art, as described, for example, in WO 2012/164270 and WO2015/150786.

An adapter useful in the selection method may comprise a tag. The tagmay be hybridized to the adapter or may be attached to the enzyme.

Suitable tags are known in the art. Examples of suitable tags include,but are not limited to, biotin, a selectable polynucleotide sequence,antibodies, antibody fragments, such as Fab and ScSv, antigens,polynucleotide binding proteins, poly histidine tails and GST tags.Biotin specifically binds to a surface coated with avidins, such asstreptavidin.

Selectable polynucleotide sequences specifically bind (i.e. hybridise)to a surface coated with complementary sequences.

The adapter and/or the tag may comprise a region that can be cut,nicked, cleaved or hydrolysed. Such a site can be designed to allow thepolynucleotides that meet the selection criteria, or polynucleotide thatdo not meet the selection criteria, to be removed from the surface,beads or column to which they are bound. Suitable sites are known in theart. Suitable sites include, but are not limited to, an RNA region, aregion comprising desthiobiotin and streptavidin, a disulphide bond, aphotocleavable region and a restriction enzyme site, or other site thatis selectively cleaved by an enzyme.

The adapter may, in addition to or instead of a tag, comprise a hiddensite for attaching a further polynucleotide and/or other molecule, suchas, for example, a protein. The adapter may, in addition to or insteadof a tag, comprise an exposed site for attaching a furtherpolynucleotide and/or other molecule, such as, for example, a protein.The further polynucleotide or other molecule, such as, for example, aprotein is used to create a selection bias.

A site for attaching a further polynucleotide may, for example, be asingle stranded region that is capable of hybridising to a complementarypolynucleotide strand or to a strand comprising or consisting ofuniversal bases, such as inosines. The complementary polynucleotidestrand may be DNA, RNA, a DNA/RNA hybrid, PNA, LNA, BNA and/or. a strandcomprising or consisting of modified bases. The modified bases may, forexample, be abasic nucleotides, such as nucleotides in which thenucleobase is replaced by —H (idSp) or —OH. The modified bases may, forexample, include one or more of 3-methyladenine, 7-methylguanine,1,N6-ethenoadenine inosine or hypoxanthine and the nucleobases may beremoved from these nucleotides using Human Alkyladenine DNA Glycosylase(hAAG). The polynucleotides may be modified to include uracil and thenucleobases removed with Uracil-DNA Glycosylase (UDG). The modifiedbases may, for example, be 2′-O-Methyl (2′OMe) and/or 2′-fluoro bases.The complementary or universal strand may be present in an adapter, suchas a Y adapter for characterising the selected polynucleotides using atransmembrane pore, e.g. the further polynucleotide may be an adapter,such as a Y adapter.

A site for attaching a molecule, may for example, be a single strandedDNA section that can bind, when exposed, to a single stranded DNAbinding protein (SSB), such as the E. coli single stranded bindingprotein.

The further polynucleotide or other molecule, such as, for example, aprotein that is used to create a selection bias may, for example, betagged, allow ligation to the polynucleotides to which the strand ishybridised, or conversely to prevent ligation to the polynucleotides towhich the strand is hybridised, allow digestion, or conversely preventdigestion. The ligation may be direct ligation, for example using aligase or indirect ligation such as using click chemistry.

The adapter may, in addition to or instead of a tag, comprise a hiddensite that can be ligated to another strand when the site becomesexposed. The adapter may, in addition to or instead of a tag, comprisean exposed site for attaching a further polynucleotide that can beligated to another strand.

The adapter may, in addition to or instead of a tag, comprise a hiddensite that allows digestion of the strand when it becomes exposed. Theadapter may, in addition to or instead of a tag, comprise an exposedsite that allows digestion of the strand when it becomes exposed.

The adapter may, in addition to or instead of a tag, contain a chemicalgroup suitable for click chemistry attachment that is hidden or exposed.

The hidden site may comprise a secondary structure such as a hairpin.For example, a hairpin could be ligated shut by the action of theenzyme, for example to prevent attachment of a sequencing adapter or toallow rolling circle amplification of a specific template.

An exposed site may be removed from polynucleotides which do not meetthe section criteria during the separation method. A hidden site istypically revealed in polynucleotides which do meet the selectioncriteria.

The adapter may be single stranded and/or double stranded. The adaptermay, for example, contain both single stranded and double strandedsections. The adapter may attach to one strand, or preferably to bothstrands of a double stranded polynucleotide. An adapter may be attachedto one or both ends of each of the multiple polynucleotides.

Defined Time Period

In step (i) of the selection method, the enzyme is allowed to move alongthe multiple polynucleotides for a defined time period. The length ofthe defined time period is based on the selection criteria, for examplethe length of the polynucleotides it is wished to select/deselect, onthe enzyme chosen as the nucleic acid handling enzyme and on thereaction conditions. It is within the routine skill of a person skilledin the art to determine a suitable defined time period. For example, theskilled person would be able to take a sample comprising apolynucleotide of the desired length and to allow an enzyme to movealong the control polynucleotide for various time periods under theconditions to be used in the method. The skilled person would then beable to determine after what time period the enzyme moves off the end ofthe polynucleotide. The defined time period can then be chosen to beless than the time taken for the enzyme to move off the end of thecontrol polynucleotide of the desired length. The selection method maythen be used to separate polynucleotides of the desired length fromshorter polynucleotides.

The defined time period may be of any length, for example from about 1second to about 14 days or longer, about 5 seconds to about 10 days,about 10 seconds to about 7 days, about 20 seconds to about 5 days,about 25 seconds to about 2 days or about 1 minute to about 1 day.

Starting Time Period

Movement of the enzyme along the multiple polynucleotides may beinitiated in any suitable way. The method of initiating movement willdepend on how the method is being carried out. In an embodiment whereone or more molecules of the enzyme are attached to the multiplepolynucleotides prior to step (i), the defined time period is typicallystarted by initiating movement of the enzyme. Movement of the enzyme maybe initiated, for example, by changing the conditions so that the enzymeis able to move. For example, a nucleotide that provides energy for theenzyme, a co-enzyme and/or a co-factor may be added to initiate movementof the enzyme. Alternative examples of ways in which movement of theenzyme may be initiated include changing the pH, temperature and/or saltconcentration, and/or pushing the enzyme over a spacer that has beenused to stall the enzyme by hybridisation of a strand behind the enzyme,e.g. by toehold displacement.

In an embodiment where the enzyme is not pre-bound to the multiplepolynucleotides prior to step (i), the defined time period is typicallystarted by contacting the multiple polynucleotides with the enzyme. Inthis embodiment, the multiple polynucleotides are contacted with theenzyme under conditions suitable for the enzyme to move along thenucleotides. For example, the enzyme is added under pH, temperature andsalt conditions amenable to movement of the enzyme. A nucleotide thatprovides energy for the enzyme and any necessary co-enzymes and/orco-factors are also typically added with the enzyme, or are alreadypresent in the sample.

In an embodiment of the invention where prior to step (i) the enzyme isallowed to bind to the multiple polynucleotides from solution, at thestart of the defined time period additional molecules of the enzyme areprevented from binding to the multiple polynucleotide. This may beachieved in any suitable way. For example, the enzymes that are notbound to the multiple polynucleotides may be sequestered by binding to acapture strand that is added to the mixture at the start of the definedtime period. In one embodiment, capture strand or another molecule thatsequesters unbound enzyme may be added together with, for example, anucleotide that provides energy for the enzyme, a co-enzyme and/or aco-factor. Alternatively or additionally, the salt concentration can beadjusted, typically by adding salt, such that rebinding of enzymes tothe polynucleotides is prevented. Alternatively or additionally, theenzyme can be closed around the polynucleotide before initiation of thedefined time period. This can be achieved, for example by ligating anadapter that contains the enzyme binding site to the ends of thestrands, then adding the enzyme in solution, closing all of the enzymes,for example with tetramethylazodicarboxamide (TMAD) and increasing thesalt concentration to prevent binding of other enzymes in solution.

An alternative to adding a capture strand may, where the multiplepolynucleotides are bound to a bead, column or surface via an adapter,to wash away any unbound enzyme and add in solution any necessary fuel,coenzymes and/or cofactors.

Free Nucleotides and Co-Factors

Movement of the enzyme can be controlled by adding or removing fueland/or co-enzymes/co-factors. Fuel is typically free nucleotides or freenucleotide analogues. The enzyme may be added to/bound to the multiplepolynucleotides in the absence of free nucleotides or free nucleotideanalogues and in the absence of any co-enzymes and/or co-factor requiredfor enzyme movement. The free nucleotides may be one or more of, but arenot limited to, adenosine monophosphate (AMP), adenosine diphosphate(ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP),guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidinemonophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate(TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridinetriphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate(CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate(cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosinemonophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosinetriphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosinediphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidinemonophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidinetriphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridinediphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidinemonophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidinetriphosphate (dCTP). The free nucleotides are preferably selected fromAMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotidesare preferably adenosine triphosphate (ATP). The enzyme cofactor is afactor that allows the polynucleotide binding protein to function. Theenzyme cofactor is preferably a divalent metal cation. The divalentmetal cation is preferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactoris most preferably Mg²⁺.

Stopping Time Period

The defined time period is stopped by inhibiting movement of the enzyme.

Movement of the enzyme may be stopped by any suitable method such as:(a) removing a nucleotide that provides energy for the enzyme, acoenzyme and/or a cofactor; (b) adding an enzyme inhibitor; (c) changingthe pH, temperature or salt concentration; and/or (d) denaturing theenzyme.

In an embodiment where the multiple polynucleotides are bound to a bead,column or surface, for example via a tag on the enzyme or a tag in anadapter, the defined time period may be stopped by washing the bead,column or surface. This may serve to remove polynucleotides that do notmeet the selection criteria. The reaction may then be restarted byadding a fuel, co-enzyme and/or co-factor in order to further separatethe multiple polynucleotides remaining on the bead, column or surface.Situations where this may be desirable are shown in the Figures.

In one embodiment, the defined time period is stopped when the amount offuel, e.g. amount of a nucleotide that provides energy for the enzyme,is depleted causing movement of the enzyme to stop. The fuel may bedepleted by movement of the enzyme along the multiple polynucleotides.Alternatively, fuel may be depleted in another way, such as by adding acompetitor enzyme, for example an enzyme with higher affinity for thefuel, to deplete the fuel and cause enzyme movement to stop.

In an embodiment where the adapter comprises a hidden tag that isrevealed when a molecule of the enzyme reaches the end of apolynucleotide, the defined time period may be stopped by contacting thereaction mixture with beads, a column or surface such that shorterpolynucleotides with the tag revealed may be removed, leaving the longerpolynucleotides that meet the selection criteria in solution.

In an embodiment where the adapter or enzyme is tagged, the multiplepolynucleotides may be bound to beads, a column or a surface at thestart of step (i). The reaction may then be stopped by washing thebeads, column or surface. This will remove any fuel, coenzymes and/orcofactors required for enzyme movement. At the same time polynucleotidesare not bound to the bead, surface or column may be separated from thosethat are bound.

In one embodiment, the defined time period is determined by the amountof fuel and/or co-factor present in the reaction conditions during theselection. The time period ends when the enzyme runs out of fuel and/orcofactor and can no longer move along the polynucleotides. It is withinthe routine skill of a person skilled in the art to determine the rateof fuel turnover by the enzyme. By controlling the amount of fuel,polynucleotides and/or enzyme present in the reaction the skilled personwould be able to control the length of time for which the enzyme will beactive and hence define a time period during which enzyme movementoccurs. Typically, in this embodiment, the amount of fuel is limited sothat the fuel is depleted within a defined time period.

Selecting

The method may be used to select multiple polynucleotides that meet theselection criteria. A subgroup of the multiple polynucleotides in asample may be selected. In the method more than one polynucleotide maybe selected, such as at least 2, at least 3, at least 4, at least 5, forexample from about 10 to about 100,000, about 20 to about 10,000, about30 to about 1,000 or about 50 to about 500 polynucleotides.

The selection method is used to distinguish polynucleotides havingdifferent physical characteristics. In one embodiment, the methodcomprises a step of separating selected polynucleotides from unselectedpolynucleotides. A number of different selections could be performed incombination. In one embodiment, the method selects polynucleotides thatcan then be separated and/or selectively modified based on theirlengths. Preferably, the method is used to select long polynucleotides.The length of the selected polynucleotides may vary depending on what isdesired. The method may be adjusted such that polynucleotides of thedesirable length are selected. For example, the method may be used toselect polynucleotides having a length of at least 1 kb, 10 kb, 50 kb,100 kb, 1,000 kb, 10,000 kb, 100,000 kb, 200,000 kb or 250,000 kb.

In one embodiment, the method may be used to distinguish damagedpolynucleotides from undamaged polynucleotides. The undamaged or damagedpolynucleotides can be selected and separated. The damaged or undamagedpolynucleotides can be selectively modified. A damaged polynucleotide isany polynucleotide which comprises an alteration in its chemicalstructure, for example, an alteration in the chemical structure of DNAor RNA. Polynucleotide damage includes a break in one strand of a doublestranded polynucleotide, one or more base missing from thepolynucleotide, or one or more chemically altered base. Thus, the methodcan be used to separate polynucleotides with nicked strands from intactpolynucleotides. A polynucleotide with a nicked strand may, in oneembodiment, be a double stranded DNA in which there is at least onebreak in one of the DNA strands. The method can be used to select intactpolynucleotides. Examples of how the method may be implemented toseparate damaged polynucleotides from undamaged polynucleotides areshown in the Figures. The Figures also show how polynucleotides ofdifferent lengths may be separated.

The method may be used to select polynucleotides that fall within aparticular “window”. For example, the method may be carried out two ormore times to select a polynucleotide within a defined range of lengths,i.e. by removing polynucleotides that are shorter than the desiredlength and then polynucleotides that are longer than the desired lengthor vice versa. Additionally, or alternatively, the method steps may becarried out two or more times to separate damaged polynucleotides fromundamaged polynucleotides and to select polynucleotides of a desiredlength. Steps (i) and (ii) of the method may be repeated once, twice,three time or more, such as four, five or six times. By repeating themethod, polynucleotide fractions may be obtained, wherein each fractioncontains polynucleotides of a different length from the other fractions.

The method selects the polynucleotides based on whether the enzymereaches the end of the polynucleotides and/or whether the enzyme unbindsfrom the polynucleotides. This selection may be achieved in any suitableway. Thus, the selection of polynucleotides may be based on certainpolynucleotides in the sample having an enzyme bound thereto at the endof the defined time period whilst other polynucleotides in the sample donot. Other examples of how the selection may be achieved include endmodification and enzyme tagging. In one embodiment, the method is usedto separate polynucleotides comprising adapters at both ends fromun-adapted polynucleotides and/or polynucleotides comprising an adapterat only one end.

End modification allows polynucleotides which have had an enzyme moveoff the end to be separated from polynucleotides which have not had anenzyme move off the end or to be selected for further processing and/orcharacterisation without being physically separated from otherpolynucleotides in the sample. In one embodiment of end modification, anadapter comprising an end signal is attached to one or both ends of eachof the multiple polynucleotides. The end signal may, for example, be aselection tag, a hidden site for attaching a further polynucleotide, anexposed site for attaching a further polynucleotide or a cap preventingdigestion by an exonuclease. Where such an end signal is used, in step(i) the selection tag is displaced, the hidden site is revealed, theexposed site is removed or the cap is removed when a molecule of theenzyme reaches the end of the polynucleotide. In step (ii), theselection tag, hidden site, exposed site or uncapped end may then beused to separate the polynucleotides.

Where the end signal is a selection tag, the multiple polynucleotidesmay be bound to a bead, column or surface via the selection tag.Displacement of the selection tag by the enzyme then results in thepolynucleotide to which the enzyme is no longer attached being releasedfrom the bead, column or surface. In this embodiment, thepolynucleotides that remain bound to the bead, column or surface afterwashing may be further separated by repeating steps (i) and (ii). Whensteps (i) and (ii) are repeated, the polynucleotides meeting theselection criteria may be those eluted from the bead, column or surface,or may be those remaining bound to the bead, column or surface. Whenonly polynucleotides meeting the selection criteria remain bound to thebead, column or surface, those polynucleotides may be recovered from thebead, column or surface, for example, by: restarting movement of theenzyme and allowing the enzyme to reach the ends of the polynucleotides;or unbinding the polynucleotides from the bead, column or surface.Unbinding may be achieved by standard methods known in the art. Forexample, the adapter may comprise a cleavage site that can be used torelease the polynucleotides.

In another embodiment that uses an end signal that is a selection tag,the selection tag may be hidden in the adapter. For example, theselection tag could be hidden by being bound to a binding partner. Forexample, if the tag is biotin, it could be bound to DNA or an avadin,such as streptavidin. When the enzyme reaches the end of apolynucleotide, the partner of the binding tag may be removed. A bead,surface or column having the binding tag partner attached thereto couldthen be used to select, or remove, polynucleotides in which the end taghas been revealed.

A hidden site that functions as an end signal may be any site, such as aDNA sequence, that enables the DNA to be ligated or attached in any wayto a further polynucleotide or to another molecule. For example, thehidden site may be a site enabling attachment to another molecule viaclick chemistry. Methods of using click chemistry attachment aredescribed in, for example, International patent application no.PCT/GB2017/051493.

Click chemistry is advantageous because it does not typically involvethe use of enzymes (Kolb et al (2001) Angew. Chem. Int. Ed. 402004-2021). Suitable example of click chemistry include, but are notlimited to a copper-free variant of the 1,3 dipolar cycloadditionreaction, where an azide reacts with an alkyne under strain, for examplein a cyclooctane ring; the reaction of an oxygen nucleophile on onelinker with an epoxide or aziridine reactive moiety on the other; andthe Staudinger ligation, where the alkyne moiety can be replaced by anaryl phosphine, resulting in a specific reaction with the azide to givean amide bond.

Preferably the click chemistry reaction is the Cu (I) catalysed 1,3dipolar cycloaddition reaction between an alkyne and an azide. In apreferred embodiment, the first group is an azide group and the secondgroup is an alkyne group. Nucleic acid bases incorporating azide andalkyne groups in preferred positions are known (for example in Kocalkaet al (2008) Chembiochem. 9(8):1280-5). Alkyne groups are availablecommercially from Berry Associates (Michigan, USA) and azide groups aresynthesised by ATDBio.

Copper free click chemistry can be used. It is fast, clean and notpoisonous towards proteins. A good example of this is maleimide oriodoacetamide linking with a cyclooctyne functional group (DIBO). Othersuitable bio-orthogonal chemistries include, but are not limited to,Staudinger chemistry, hydrazine or hydrazide/aldehyde or ketone reagents(HyNic+4FB chemistry, including all Solulink™ reagents), Diels-Alderreagent pairs and boronic acid/salicyhydroxamate reagents.

Preferably the reactive groups are azide and hexynl groups such as3AzideN and 5′-hexynl-G. Preferred pairs of non-covalent reactive groupsinclude, but are not limited to, (i) Ni-NTA and polyhistidine, such as6×His, and (ii) cyclodextrin and adamantine. The hidden site may behidden, or protected, in any way. For example, a molecule may be used tohide, or occlude, a click reactive group. Movement of the enzyme mayremove the molecule and reveal the reactive group. Any suitable moleculemay be used. For example, pyrene may be used to stack with the DBCO. Ifthe hidden site comprises a Ni-NTA group (which can attach topolyhistidine, such as 6×His), the hidden site may be protected withpolyhistidine, such as 6×His, in the same target polynucleotide and viceversa, i.e. the hidden site may comprise polyhistidine, such as 6×His,and may be protected by Ni-NTA groups. If the hidden site comprisescyclodextrin (which can attach to amantadine in the subsequentpolynucleotide), the hidden site may be protected by amantadine in thesame target polynucleotide or vice versa. The hidden site and theprotecting molecule may be present on opposite strands of an adapter andhence on opposite stands of a polynucleotide to which the adapter isattached. Separation of the strands by the enzyme may then separate theprotecting molecule from the hidden site and reveal the hidden site.

The hidden site may be protected by hybridisaton to a protectingpolynucleotide. The protecting polynucleotide may be removed when theenzyme reaches the end of the polynucleotide. In one example, theprotecting polynucleotide may prevent the action of a single strandligase on the hidden site. Release of the protecting polynucleotidewould reveal the hidden site as a substrate for the ligase.

The end signal may be created by movement of the enzyme over the adapterat the end of the polynucleotide altering the secondary or tertiarystructure of the polynucleotide. For example, movement of the enzyme mayunwind a hairpin or quadruplex to alter the ability of the adapter tomove a further polynucleotide.

In one embodiment, in step (ii) of the method polynucleotides to whichthe enzyme remains bound are separated from polynucleotides to which theenzyme is not bound. One way of achieving this is to use a taggedenzyme.

In one embodiment, a tagged enzyme is attached to an adapter, such thatat the start of step (i) of the method, one molecule of enzyme movesalong the polynucleotide, or one molecule of the enzyme moves along eachstrand of the polynucleotide in opposite directions. If the taggedenzyme remains on the polynucleotide at the end of the defined timeperiod, the polynucleotide is tagged via the tag on the enzyme and canbe separated from shorter polynucleotides on which no enzyme found.

In another embodiment, where multiple molecules of the tagged enzyme arebound to the multiple polynucleotides, all of the molecules of thetagged enzyme present on the polynucleotide move along thepolynucleotides. If any molecules of tagged enzyme remain on thepolynucleotide at the end of the defined time period, the polynucleotideis tagged via the tag on the enzyme and can be separated from shorterpolynucleotides from which all molecules of the tagged enzyme have movedoff.

In one embodiment, the tag on the enzyme may be used to bind the enzymeto a bead, column or surface. The enzyme may be bound to the bead,column or surface prior to carrying out step (i) of the method.Alternatively, the bead, column or surface may be used in step (ii) ofthe method to bind polynucleotides which retain a tagged enzyme. In anembodiment where the enzyme is bound to beads, a column or a surfaceprior to step (i), the defined time period of step (i) can be terminatedby quenching the solution or by removing the beads, column or surfacefrom the reaction solution containing the necessary fuel, co-enzymesand/or co-factors necessary for enzyme movement. Steps (i) and (ii) canbe repeated to collect multiple fractions of different lengths and/or toselect polynucleotides of a certain length and/or to separate outdamaged and undamaged polynucleotides.

In one embodiment, the adapter may comprise a sequence that is capableof hybridising to a complementary polynucleotide strand, whichcomplementary strand is tagged, for example, with biotin ordesthiobiotin. The complementary strand may comprise part of theadapter, and/or may be pre-bound to a bead, column or surface. Theadapter may be used to bind the multiple polynucleotides to a bead,column or surface. Movement of the enzyme in step (i) then displacespolynucleotides that are too short to meet the selection criteria fromthe bead, column or surface. The polynucleotides remaining bound to thebeads, column or surface may be damaged or undamaged polynucleotides.Steps (i) and (ii) may be repeated to select only for undamaged strands.The damaged strands will remain bound to the bead, column or surfacewhilst the undamaged strands will be released.

In one particular embodiment, the adapter may be ligated to, or comprisea DNA containing a binding site and a priming site for a stranddisplacing DNA polymerase. Movement of the polymerase alongpolynucleotides which are sufficiently short for the polymerase to reachthe end in the defined time period may be used to displace abiotinylated strand polynucleotide. Alternatively, the polymerase may betagged.

In methods which use a selection tag, an extraction medium is typicallyused to separate tagged polynucleotides from untagged polynucleotides.Any suitable extraction medium may be used. Examples of suitableextraction media include beads, columns and surfaces. In one embodiment,the surface may be a membrane comprising a transmembrane pore.

The enzyme or adapter can be pre-bound to an extraction medium. Use ofan extraction medium enables washing to: quench by flushing; removeunwanted polynucleotides that do not meet the selection criteria; and/orclute desired polynucleotides that do meet the selection criteria.

Hence, the defined time period may be stopped by flushing a bead, columnor surface and/or polynucleotides not bound to the bead, column orsurface may be removed or eluted by washing the bead, column or surfaceat the end of the defined time period.

In one embodiment, the polynucleotides that remain bound to the bead,column or surface after washing are further separated by repeating steps(i) and (ii).

Polynucleotides that remain bound to the bead, column or surface may berecovered from the bead, column or surface by any suitable method, suchas: restarting movement of the enzyme and allowing the enzyme to reachthe ends of the polynucleotides; unbinding the enzyme from the bead,column or surface; or unbinding the enzyme from the polynucleotides.

One example of an exposed site in an adapter is an overhang that allowsefficient ligation to a further polynucleotide such as a sequencingadapter. If the enzyme reaches the end of the strand in the defined timeperiod (i.e. when the strand is short), the adapter may fold into aconformation in which the overhang for ligation is no longer available.This will allow selection of strands of a desired length, but not toselect for un-nicked strands. Alternatively, the enzyme may displace thestrand containing the specific overhang. This alternative isparticularly useful for adding sequencing adapters with a 5′ overhang tothe selected polynucleotides.

The end signal may, in one embodiment, be a cap preventing digestion byan exonuclease. Digestion by an exonuclease may be prevented by, forexample, including one or more of the following in the adapter:phosphorothioate (PS) bonds at the 5′ end and/or 3′ end, typically atleast 3 PS bonds at the 5′ end and/or 3′ end; 2′-O-Methyl (2′OMe)modified nucleotides at the 5′ end and/or 3′ end; 2-fluoro bases at the5′end and/or 3′ end: inverted dT and ddT at the 5′ end and/or 3′ end;phosphorylated 5′ and/or 3′ nucleotides; a spacer, such as aphosphoramidite C3 Spacer, at the 5′ end and/or 3′ end.

Selected DNA may be used for any purpose. Many platforms requireintact/long DNA polynucleotides or indeed only short polynucleotidefragments. For example, the polynucleotides may be characterised, suchas sequenced using any suitable sequencing method. Typically, any highthroughput sequencing method may be used.

In one particular embodiment, the method may be used to select targetpolynucleotides for delivery to a transmembrane pore.

To achieve this, an adapter may be attached to each of the multiplepolynucleotides, wherein a second nucleic acid handling enzyme isattached to the adapter and is stalled on the adapter such that it doesnot move along the polynucleotide in step (i).

Alternatively, step (ii) may comprise: binding the hidden or exposedsite to an adapter comprising a single stranded leader sequence andoptionally a membrane anchor or a transmembrane pore anchor, wherein asecond nucleic acid handling enzyme is attached to the adapter and isstalled on the adapter; and bringing the sample into contact with atransmembrane pore.

Where the selected polynucleotides are bound to a bead, thepolynucleotide may be contacted with the pore whilst still bound to thebeads. The heads may be used to facilitate delivery of the selectedpolynucleotides to the pore, for example as disclosed in WO 2016/059375.

Where the polynucleotides are bound to a surface, the surface may be amembrane comprising the pore. The selection tag may, in this embodiment,be a membrane anchor, such as cholesterol. The membrane anchor may, forexample, be bound to the enzyme, may be hidden in the adapter andrevealed by enzyme reaching end, or may be present in the adapter and acleavage site by which the adapter may be removed may be revealed by theenzyme reaching the end.

The membrane anchor may be a polypeptide anchor and/or a hydrophobicanchor that can be inserted into the membrane. The hydrophobic anchor ispreferably a lipid, fatty acid, sterol, carbon nanotube, polypeptide,protein or amino acid, for example cholesterol, palmitate or tocopherol.The anchor may comprise thiol, biotin or a surfactant.

In one aspect the anchor may be biotin (for binding to streptavidin),amylose (for binding to maltose binding protein or a fusion protein),Ni-NTA (for binding to poly-histidine or poly-histidine tagged proteins)or peptides (such as an antigen).

The anchor may comprise a linker, or 2, 3, 4 or more linkers. Preferredlinkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. The adapter mayhybridise to a complementary sequence on a circular polynucleotidelinker. The one or more anchors or one or more linkers may comprise acomponent that can be cut or broken down, such as a restriction site ora photolabile group. The linker may be functionalised with maleimidegroups to attach to cysteine residues in proteins. Suitable linkers aredescribed in WO 2010/086602.

The anchor is preferably cholesterol or a fatty acyl chain. For example,any fatty acyl chain having a length of from 6 to 30 carbon atom, suchas hexadecanoic acid, may be used.

Examples of suitable anchors and methods of attaching anchors toadapters are disclosed in WO 2012/164270.

A method of characterising a polynucleotide is provided. Thecharacterisation method comprises:

-   -   (i) carrying out a selection method as described herein;    -   (ii) contacting a transmembrane pore with the selected        polynucleotides;    -   (iii) applying a potential difference across the transmembrane        pore; and    -   (iv) taking one or more measurements which are indicative of one        or more characteristics of a polynucleotide moving with respect        to the transmembrane pore and thereby characterising the        polynucleotide.

The one or more characteristics may be selected from (i) the length ofthe polynucleotide, (ii) the identity of the polynucleotide, (iii) thesequence of the polynucleotide, (iv) the secondary structure of thepolynucleotide and (v) whether or not the polynucleotide is modified.

The method characterisation typically comprises measuring the currentpassing through the transmembrane pore as the polynucleotide moves withrespect to the transmembrane pore.

Transmembrane pores and nucleic acid handling enzymes are known in theart. Suitable membranes and devices are also known, as are methods foranalysing the current signal to determine sequence and othercharacteristics of the polynucleotides.

Kits

A kit for separating polynucleotides is provided comprising one or moreadapters for use in the separation method as defined herein; and any oneor more, including any combination, of the following components:

-   -   an extraction medium;    -   a nucleic acid handling enzyme;    -   a nucleotide that provides energy for the enzyme, an enzyme        cofactor and/or a coenzyme;    -   a solution comprising fuel and/or cofactor for the nucleic acid        handling enzyme;    -   wash solution, which does not contain fuel and/or cofactor for        the nucleic acid handling enzyme;    -   a site specific endonuclease; and/or    -   a sequencing adapter.

The adapter may, for example, be an adapter comprising an end signal.The end signal may, for example, be a selection tag, a hidden site forattaching a further polynucleotide, an exposed site for attaching afurther polynucleotide or a cap preventing digestion by an exonuclease.

The selection tag may be hidden in the adapter. For example, theselection tag could be hidden by being bound to a binding partner. Forexample, if the tag is biotin, it could be bound to DNA or an avadin,such as streptavidin. The selection tag may be a membrane anchor, suchas cholesterol. The selection tag may, for example, be biotin (forbinding to streptavidin), amylose (for binding to maltose bindingprotein or a fusion protein), Ni-NTA (for binding to poly-histidine orpoly-histidine tagged proteins) or peptides (such as an antigen). Thekit may further comprise a molecule that binds to the selection tag. Themolecule that binds to the selection tag may be coupled to theextraction medium.

The hidden site may be any site, such as a DNA sequence, that enablesthe DNA to be ligated or attached in any way to a further polynucleotideor to another molecule. The adapter may, for example, have a singlestranded overhang that can hybridise to the complementary sequence in afurther polynucleotide. The overhang may have a length of from about 4to about 15 nucleotides, such as about 6, 8, 10 or 12 nucleotides. Thehidden site may be a site enabling attachment to another molecule viaclick chemistry. Suitable sites are disclosed above.

The hidden site may be hidden, or protected, for example, by a moleculethat occludes the click reactive group. For example, pyrene may be usedto stack with the DBCO. If the hidden site comprises a Ni-NTA group(which can attach to polyhistidine, such as 6×His), the hidden site maybe protected with polyhistidine, such as 6×His, in the same targetpolynucleotide and vice versa, i.e. the hidden site may comprisepolyhistidine, such as 6×His, and may be protected by Ni-NTA groups. Ifthe hidden site comprises cyclodextrin (which can attach to amantadinein the subsequent polynucleotide), the hidden site may be protected byamantadine in the same target polynucleotide or vice versa. The adaptermay comprise a hidden site on one strand and a protecting molecule onthe opposite strand.

The hidden site may be protected by hybridisaton to a protectingpolynucleotide. The protecting polynucleotide may be removed when theenzyme reaches the end of the polynucleotide. The protectingpolynucleotide may for example be at least partially complementary to asingle stranded overhang sequence in the adapter, for example theprotecting polynucleotide may have from about 70% to 100% identity tothe overhang sequence, such as about 75%, 80%, 80% or 95% identity. Inone example, the protecting polynucleotide may prevent the action of asingle strand ligase on the hidden site. Release of the protectingpolynucleotide would reveal the hidden site as a substrate for theligase.

The end signal may be a secondary or tertiary structure such as hairpinor quadruplex that can be removed, such as unwound, by movement of theenzyme. One example of an exposed site in an adapter is an overhang thatallows efficient ligation to a further polynucleotide such as asequencing adapter. If the enzyme reaches the end of the strand in thedefined time period (i.e. when the strand is short), the adapter mayfold into a conformation in which the overhang for ligation is no longeravailable. This will allow selection of strands of a desired length, butnot to select for un-nicked strands. Alternatively, the enzyme maydisplace the strand containing the specific overhang. This alternativeis particularly useful for adding sequencing adapters with a 5′ overhangto the selected polynucleotides.

The end signal may, in one embodiment, be a cap preventing digestion byan exonuclease. Digestion by an exonuclease may be prevented by, forexample, including one or more of the following in the adapter:phosphorothioate (PS) bonds at le 5′ end and/or 3′ end, typically atleast 3 PS bonds at the 5′ end and/or 3′ end 2′-O-Methyl (2′OMe)modified nucleotides at the 5′ end and/or 3′ end; 2′-fluoro bases;inverted dT and ddT at the 5′ end and/or 3′ end; phosphorylated 5′and/or 3′ nucleotides; a spacer, such as a phosphoramidite C3 Spacer, atthe 5′ end and/or 3′ end.

The nucleic acid handling enzyme may be any of the enzymes discussedabove.

The extraction medium may be beads, a column or surface. The nucleicacid enzyme or adapter may be tagged. The tag (selection tag) is capableof binding to extraction medium. Extraction medium may be modified tofacilitate binding of the tag. For example, coated with biotin where thetag is a strep tag.

In one embodiment, the adapter may comprise a sequence that is capableof hybridising to a complementary polynucleotide strand, whichcomplementary strand is tagged, for example, with biotin ordesthiobiotin. The complementary strand may comprise part of theadapter, and/or may be pre-bound to a bead, column or surface.

In one particular embodiment, the adapter may be ligated to, or comprisea DNA containing a binding site and a priming site for a stranddisplacing DNA polymerase.

The enzyme or adapter in the kit can be pre-bound to an extractionmedium.

The hidden or exposed site may after separation of the polynucleotidesbe bound to one or more sequencing adapter, or otherwise modified foruse in a method of characterisation or any other method requiring thepolynucleotide(s). The kit may include one or more sequencing adapters.Suitable sequencing adapters are known in the art and others aredescribed herein. The adapter comprising an end signal may be asequencing adapter.

The following Examples illustrate the invention.

Example 1

This Example demonstrates that a polynucleotide may be selected based onits length using a DNA translocase. The method used corresponds to theembodiment outlined in FIG. 1.

Materials and Methods Preparation of the Size Selection Adapter

The size selection adapter comprises a top strand (SEQ ID NO: 1), abottom strand (SEQ ID NO: 2) and a blocker strand (SEQ ID NO: 3)annealed together at 4.72 μM, 5.66 μM and 5.66 μM respectively in 50 mMHEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at 2° C. perminute. 40 μL of annealed DNA was added to 360 μL of a 4 μM DNA helicase(Dda) containing a strep-SUMO tag (SEQ ID NO: 4 below). The helicase wasbound to the size selection adapter for 45 minutes at room temperature,and 5 L of 8.1 mM Diamide (SIGMA-ALDRICH) was added. The sample wasincubated at 35° C. for 60 minutes. 40 μL of 5 M NaCl and 15 μL ofNuclease free water (Ambion™) was added to the sample. The sample wasincubated for 30 minutes at room temperature. The sample was purified onan HQ10 column, and the free annealed DNA and helicase bound annealedDNA were separated into different fractions. The helicase bound DNA isreferred to below as the ‘size selection adapter’.

Ligation of Size Selection Adapter to DNA

3608 ng of end-repaired and dA-tailed 3.6 kb DNA (SEQ ID NO: 5) wasligated for 60 minutes at room temperature in 300 μL with 150 μL of 35nM size selection adapter, 15 μL of T4 DNA Ligase (NEB), 60 μL ofligation buffer (50 mM Tris pH8.0, 50 mM MgCl2, 10 mM ATPγS, 32% PEG8000) and 71 L of nuclease free water. This is referred to below as the“ligation mixture”.

Binding of DNA Ligated to Size Selection Adapter to Beads

50 L of MagStrep “Type 3” beads (IBA) were washed twice with 150 L of 50mM Tris-HCl (pH 8.0 @ 4 C), 150 mM NaCl, 1 mM EDTA. The beads wereresuspended in 150 L of 50 mM Tris-HCl pH8.0 @ 4 C, 20 mM NaCl, andadded to the ligation mixture.

The beads were pelleted on a magnetic rack. The supernatant was removed.The beads were washed twice with 150 μL of 50 mM Tris-HCl (pH 8.0 @ 4 C)150 mM NaCl, 1 mM EDTA. The beads were re-suspended in 150 μL of 50 mMTris-HCl pH8.0 @ 4 C, 20 mM NaCl.

Movement of Helicase

The sample was split into twelve 10 μL fractions. The beads werepelleted and the buffer removed. Fuel was added to initiate helicasemovement by re-suspending the beads in 5 μL of ATP buffer (1 mM MgCl2,10 mM ATP, 150 mM NaCl and 50 mM Tris pH8.0). The sample was left toincubate with the ATP buffer for 15, 30, 60, 75, 90, 120, 300, 600, 900,1800 or 3600 seconds. At the end of the defined time period, helicasemovement was stopped by pelleting the beads and adding 5 L of 0.5M EDTA(SIGMA) to the sample and mixed thoroughly. The beads were pelleted andthe 10 L supernatant was removed to a separate tube. A 0 second samplewas made in which no ATP buffer was added to the sample, instead 10 of0.5 M EDTA was added.

Determining Time Point at which 3.6 kb DNA was Released from the Beads

2 μL of purple loading dye (NEB) was added to the supernatant and mixedthoroughly. This is referred to as the ‘eluted from beads’ sample.

The bead pellet was re-suspended in 10 μL of TBA XT Biotin Elutionbuffer. Then 2 L of purple loading dye (NEB) was added to the suspensionand mixed thoroughly. This sample is referred to as the ‘left on beads’sample.

The samples were loaded onto a 4-20% TBE gel, for 90 minutes at 180 V.Stained in SYBR gold nucleic acid gel stain (Thermofisher) and imaged onthe UV gel doc it (GelDoc-It Imaging Sustems (UVP)).

Results

The results indicate that the 3.6 kb analyte is released from the beadafter a defined time period of 31-60 seconds. This indicates that thehelicase is translocating along the DNA at a rate of 60-116 base pairsper second in these buffer conditions at this temperature. When thehelicase reaches the end of the strand, the strand is released from thebead. This Example shows that DNA of desirable sizes could be separatedfrom and/or characterised independently from or preferentially to DNA ofother sizes using this method; the helicase would reach the end of thedifferently sized fragments after a different amount of time, and thesefractions could be collected separately to collect DNA of differentlengths. The helicase could be slowed down or sped up with lower orhigher ATP concentrations, to allow a higher degree of separation, or tofacilitate faster separation of very long strands.

Some of the 3.6 kb is not released from the bead after 60 seconds, thiscould indicate that the helicase has encountered some sort of DNA damageon the strand, and has become paused. This indicates that this methodcould be used for the separation of damaged from un-damaged DNA.

Example 2

This Example describes a method for selectively characterisingpolynucleotides their lengths using a tagged DNA translocase to selectpolynucleotides for characterisation. The method used corresponds to theembodiment outlined in FIG. 1.

Materials and Methods Preparation of the Size Selection Adapter

The size selection adapter comprises a top strand (SEQ ID NO: 1), abottom strand (SEQ ID NO: 2) and a blocker strand (SEQ ID NO: 6)annealed together at 4.72 μM, 5.6 μM and 5.6 μM respectively in 50 mMHEPES pH 8, 100 mM potassium acetate, 1 mM EDTA from 95° C. to 22° C. at2° C. per minute. 360 μL of a 1 μM DNA helicase (Dda) containing astrep-SUMO tag (ID4 below), in 50 mM HEPES pH 8, 100 mM potassiumacetate. 1 mM EDTA buffer was added to 40 μL of the annealed DNA. Thehelicase was bound to the size selection adapter for 45 minutes at roomtemperature, and 5 μL of 8.1 mM Diamide (SIGMA-ALDRICH) was added. Thesample was incubated at 35° C. for 60 minutes. 40 μL of 5 M NaCl and 15μL of nuclease free water were added, and the sample was left for 30minutes at room temperature. The sample was purified on an HQ10 column.The helicase bound annealed DNA was separated from the unbound DNA. Thehelicase bound DNA is referred to as the ‘size selection adapter’.

Preparation of Lambda Libraries of Different Sizes

A <1 kb library was prepared using NEB DNA fragmentase, following themanufacturer's guidelines. Libraries of ˜6 kb, ˜10 kb and ˜20 kb wereprepared separately using Covaris G-tube, following the manufacturer'sguidelines. A ˜48.5 kb library was prepared using un-fragmented lambdaDNA. Briefly, the Lambda genomic DNA from NEB (N3013) was diluted to theconcentration specified by the manufacturer in nuclease free water. TheDNA was heated to 65° C. for 5 minutes and then placed on ice. The DNAwas treated following the manufacturer's guidelines to achieve librarieswith median lengths previously specified. The DNA was then end repairedand dA tailed using NEBNext Ultra II end repair and dA tailing kit(E7546) following the manufacturer's guidelines. The sample was cleanedusing AMPure XP SPRI beads following the manufacturer's guidelines. Thelibraries were eluted in TE buffer and stored at 4° C.

Preparation of the Mixed Library

138 ng of <1 kb library, 1649 ng of 6 kb library, 2762 ng of 10 kblibrary, 5565 ng of 20 kb library and 14001 ng of 48.5 kb library weremixed together. This is referred to as the ‘mixed DNA library’.

Binding the Adapter to Beads

130 L of IBA Type 3 beads were washed twice with 200 L of 50 mM Tris-HC(pH 8.0 @ 4° C.), 150 mM NaCl, 1 mM EDTA. The size selection adapter wasbound to the beads at 4° C. overnight. The beads were washed twice with300 L of 50 mM Tris-HCl (pH 8.0 @ 4° C.), 150 mM NaCl, 1 mM EDTA. Thebeads were pelleted on a magnet.

Binding the Mixed Library to Beads Via the Adapter

The buffer was replaced with 55 μL of the 300 ng/μL mixed DNA library.10 L of T4 DNA Ligase (NEB) and 20 μL of ligation buffer (50 mM TrispH8.0, 25 mM MgCl2, 5 mM ATPγS, 32% PEG 8000 were added beforeincubating overnight at 4° C.

Movement of Helicase

The sample was split into 5, 20 μL volumes. The beads were bound to themagnet and washed with 120 μL of 10 mM Tris-HCl (pH 8.0 @ 4° C.), 150 mMNaCl, 1 mM EDTA. The beads were re-suspended in 25 μL of 25 mM HEPESpH8.0, 500 mM KCl. To four of the samples 25 μL of 10 mM Tris-HCl (pH8.0 @ 4° C.), 500 mM NaCl, 20 mM ATP and 2 mM MgCl₂ was added. Thesamples were left for 20, 40, 120 or 240 seconds. 25 μL 0.5 M EDTA wasadded after the defined time period. The beads were pelleted and washedwith 120 L of 50 mM Tris-HCl (pH 8.0 @ 4° C.), 2 M NaCl, 1 mM EDTA. Thebeads were pelleted and washed twice with 120 L of 50 mM Tris-HCl (pH8.0 @ 4° C.), 150 mM NaCl, 1 mM EDTA.

Addition of Sequencing Adapters

The beads were re-suspended in 20 μL of 10 mM Tris-HCl (pH 8.0 @ 4° C.),20 mM NaCl. 10 μL of barcode adapter mix (BAM) from Oxford Nanoporesequencing kit EXP-NBD103 and 30 L of Blunt/TA Ligase Master Mix (NEB)were added, and the samples incubated for 10 minutes at roomtemperature.

Removal of Remaining DNA from Beads

To remove the remaining DNA from the beads, the helicase was reactivatedby adding 60 μL RBF (running buffer with fuel mix) from Oxford Nanoporesequencing kit SQK-LSK108 and incubating the samples for 5 minutes atroom temperature. This allowed the helicase bound to the beads to runoff the end of the DNAs to which it was bound, and release the DNA.

Sequencing

To clean up the DNS, 60 μL AMPure XP SPRI beads was added and thesamples incubated for 10 minutes at room temperature. The sample waswashed twice with 120 μL of ABB from Oxford Nanopore sequencing kitSQK-LSK108. The sample was eluted in 21.5 μL of 10 mM Tris-HCl (pH 8.0 @4° C.), 20 mM NaCl for 10 minutes. 16 μL of elution buffer (ELB) fromOxford Nanopore sequencing kit SQK-LSK108 was added followed by 37.5 Lof RBF from Oxford Nanopore sequencing kit SQK-LSK108. 75 μL of thissequencing mix was then added to the Oxford Nanopore Minion, using theSpotOn Flowcell Port. The experiment was run at −180 mV andhelicase-controlled DNA movement was monitored.

Results

The results shown in FIG. 9A indicate that strands with a duration of <5seconds are depleted when the sample is incubated with ATP for 240seconds. The results in FIG. 9B indicate that the shorter strands aredepleted more with a longer incubation in ATP, whilst the longer strandsare not depleted.

Table 1 shows the percentages of strands binned into categories of lessthan 3000 kilobases, 3000-8000 kilobases, and greater than 8000kilobases for the initial control sample (0 s incubation) and thelibraries prepared from the 120 s and 240 s helicase incubations. Thedata shows a reduction in strands in the <3000 kb range, and an increasein the strands in 3000-8000 and >8000 ranges, for the libraries preparedfrom 120 s and 240 s incubation times.

TABLE 1 Values from FIG. 9 Incubation Number of % <3000 % 3000-8000% >8000 time (s) strands kilobases kilobases kilobases 0 (control) 134253.7 34.4 12.0 120 1357 45.2 42.4 12.4 240 1344 35.6 48.4 16.1

1. A method for selecting polynucleotides, the method comprising: (i)allowing a nucleic acid handling enzyme to move along multiplepolynucleotides in a sample for a defined time period, wherein theenzyme is loaded onto each of the multiple polynucleotides and whereinone or more molecule of the enzyme moves along each of the multiplepolynucleotides; and (ii) selecting polynucleotides based on whether ornot the enzyme reaches the end of and/or unbinds from thepolynucleotides in the defined time period. 2-4. (canceled)
 5. A methodaccording to claim 1, wherein the method comprises an initial step ofattaching an adapter to one or both ends of the multiple polynucleotidesand then binding the enzyme to the adapter or wherein the methodcomprises an initial step of attaching an adapter having the enzymepre-bound thereto to one or both ends of each of the multiplepolynucleotides under conditions where the enzyme does not move alongthe polynucleotides.
 6. (canceled)
 7. A method according to claim 1,wherein additional molecules of the enzyme are prevented from binding tothe multiple polynucleotides in step (i), optionally wherein a capturestrand which binds to the enzyme is added to the sample in step (i). 8.(canceled)
 9. A method according to claim 1, wherein the defined timeperiod is started by initiating movement of the enzyme, optionally by(a) adding a nucleotide that provides energy for the enzyme, a coenzymeand/or a cofactor, and wherein the defined time period is stopped byinhibiting movement of the enzyme, optionally by (a) removing anucleotide that provides energy for the enzyme, a coenzyme and/or acofactor; (b) adding an enzyme inhibitor; (c) changing the pH,temperature or salt concentration; and/or (d) denaturing the enzyme.10-12. (canceled)
 13. A method according to claim 1, wherein at thestart of step (i) only one molecule of the enzyme is bound to each ofthe multiple polynucleotides; or wherein at the start of step (i)multiple molecules of the enzyme are bound to each of the multiplepolynucleotides; or wherein at the start of step (i) no molecules of theenzyme are bound to any of the multiple polynucleotides and the definedtime period is started by contacting the multiple polynucleotides withthe enzyme. 14-17. (canceled)
 18. A method according to claim 1, whereinthe method comprises separating the selected polynucleotides fromunselected polynucleotides.
 19. (canceled)
 20. A method according toclaim 1, wherein an adapter comprising an end signal is attached to oneor both ends of each of the multiple polynucleotides and wherein the endsignal is a selection tag, a hidden site for attaching a furtherpolynucleotide, an exposed site for attaching a further polynucleotideor a cap preventing digestion by an exonuclease; and wherein in step (i)the selection tag is displaced, the hidden site is revealed, the exposedsite is removed or the cap is removed when a molecule of the enzymereaches the end of the polynucleotide; and in step (ii) the selectiontag, hidden site, exposed site or uncapped end is used to separate thepolynucleotides.
 21. (canceled)
 22. A method according to claim 1,wherein the multiple polynucleotides are bound to a bead, column orsurface via a selection tag and displacement of the selection tag by theenzyme results in the polynucleotide to which the enzyme is no longerattached being released from the bead, column or surface, optionallywherein the surface is a membrane comprising a transmembrane pore.23-29. (canceled)
 30. A method according to claim 1, wherein in step(ii) polynucleotides to which the enzyme remains bound are separatedfrom polynucleotides to which the enzyme is not bound. 31-33. (canceled)34. A method according to claim 1, wherein the selected polynucleotidesare longer or shorter than the unselected polynucleotides, or whereinthe selected polynucleotides are damaged and the selectedpolynucleotides are undamaged, or the selected polynucleotides areundamaged and the selected polynucleotides are damaged, or wherein theselected polynucleotides are selectively modified or characterised.35-36. (canceled)
 37. A method according to claim 1, whereinpolynucleotides with nicked strands are separated from intactpolynucleotides, and selectively modified polynucleotides are nicked andunmodified polynucleotides are intact, or the selectively modifiedpolynucleotides are intact and the unmodified polynucleotides arenicked.
 38. A method according to claim 1, wherein: in step (i) thedefined time period is started by adding a capture strand which binds tothe enzyme to prevent additional molecules of the enzyme binding to themultiple polynucleotides; and in step (ii) polynucleotides to which theenzyme remains bound are separated from polynucleotides to which theenzyme is not bound.
 39. (canceled)
 40. A method according to claim 1,wherein an adapter is attached to each of the multiple polynucleotides,wherein a second nucleic acid handling enzyme is attached to the adapterand is stalled on the adapter such that it does not move along thepolynucleotide in step (i).
 41. A method according to claim 1, whereinstep (ii) comprises: binding a hidden or exposed site to an adaptercomprising a single stranded leader sequence and optionally a membraneanchor or a transmembrane pore anchor, wherein a second nucleic acidhandling enzyme is attached to the adapter and is stalled on theadapter; and bringing the sample into contact with a transmembrane pore.42. A method according to claim 1, wherein the enzyme is a translocase,a helicase, a polymerase or an exonuclease.
 43. A method according toclaim 1, wherein the polynucleotides are DNA, RNA and/or DNA/RNAhybrids.
 44. (canceled)
 45. A method according to claim 1, wherein thesample: (a) comprises the products of a PCR reaction; (b) is a DNAlibrary; (c) comprises genomic DNA; (d) comprises the products of aendonuclease digestion.
 46. A method according to claim 1, furthercomprising one or more of the following additional steps: selectingpolynucleotides of a desired length; selecting undamagedpolynucleotides; selecting intact polynucleotides; characterisingpolynucleotides of a desired length, undamaged polynucleotides and/orintact polynucleotides; sequencing polynucleotides of a desired length,undamaged polynucleotides and/or intact polynucleotides; removingprimers or adapters from polynucleotides of a desired length, undamagedpolynucleotides and/or intact polynucleotides; and genotyping, DNAfingerprinting or profiling using polynucleotides of a desired length,undamaged polynucleotides and/or intact polynucleotides.
 47. A method ofcharacterising a polynucleotide, the method comprising: (i) carrying outa method according to claim 1; (ii) contacting a transmembrane pore withthe selected polynucleotides; (iii) applying a potential differenceacross the transmembrane pore; and (iv) taking one or more measurementswhich are indicative of one or more characteristics of a polynucleotidemoving with respect to the transmembrane pore and thereby characterisingthe polynucleotide.
 48. A method of characterising a polynucleotide, themethod comprising: (i) carrying out a method according to claim 1; (ii)contacting a transmembrane pore with the selected polynucleotides; (iii)applying a potential difference across the transmembrane pore; and (iv)taking one or more measurements which are indicative of one or morecharacteristics of a polynucleotide moving with respect to thetransmembrane pore and thereby characterising the polynucleotide,wherein the method further comprises measuring the current passingthrough the transmembrane pore as the polynucleotide moves with respectto the transmembrane pore. 49-53. (canceled)